Phenotypic screening (A.K.A. classical pharmacology) has been historically used in drug discovery. While technological developments have made the prevalence of target-based screening more popular, statistical analysis shows that a disproportionate number of first-in-class drugs with novel mechanisms of action come from phenotypic screening. Cambridge Healthtech Institute’s 3rd Annual Phenotypic Drug Discovery meeting will address the advantages of phenotypic screening vs. target-based screening, and focus on assay development, selection of physiologically relevant models, and subsequent target identification and validation.

Preclinical in vitro models that better represent the complexity of an in situ human tumor are being sought as part of the IMI project PREDECT (www.predect.eu). It is hoped that better recapitulation of the clinical situation, preclinically, will greatly improve target validation and ultimately get better drugs to patients. As part of PREDECT we have been investigating the use of organotypic slice culture for cultivation of tumor material ex vivo.

The tumor microenvironment contributes to cancer progression, metastasis and drug resistance. A multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix was adapted into a robust and reliable 384- and 1,536-multi-well high-throughput screening assay that reproduces the human ovarian cancer metastatic microenvironment. The identified inhibitors were validated using multiple cells and independent in vitro and in vivo secondary assays. These assays specifically investigated the effect of the compounds on ovarian cancer cell adhesion, invasion, proliferation and metastasis to the peritoneal microenvironment. Collectively, these findings show that a 3D organotypic culture can be adapted for high-throughput screening.

We recently developed a human neural cell culture model of Alzheimer’s disease (AD) based on a three-dimensional (3D) cell culture system. This unique cellular AD model recapitulated key events of the pathogenic cascade of this disease, including β-amyloid plaques and neurofibrillary tangles. In this talk, I will present recent updates on our 3D culture model and discuss challenges and prospects.

Organoid cultures have emerged as a powerful tool for understanding adult stem cell biology in a wide range of tissues. However, conventional organoid methodology presents unique challenges in terms of accurate quantification and retrieval of samples. Bioengineered microraft arrays address these challenges by providing a high-throughput platform for long-term clonal tracking and retrieval of organoids. As proof of concept, we use microraft arrays to study stem cell-niche interaction and probe the molecular characteristics of organoid phenotypes.

Many primary cell types, including PDX models, are not readily grown on conventional 2D tissue culture substrates. We describe our ongoing efforts using customizable, biocompatible hydrogels to enable the extended laboratory culture of cancer PDXs and other primary cells in 3D. This platform permits the use of these desirable cell lines for laboratory manipulation and drug testing in miniaturizable formats.

11:40 3D Brain Models to Study Neurotoxicity and Neurodegeneration

Lena Smirnova, Ph.D., Research Associate, Johns Hopkins Center for Alternatives to Animal Testing, Johns Hopkins University Bloomberg School of Public Health

The increasing incidence of neurodevelopmental disorders and lack of cure for neurodegenerative diseases such as Parkinson’s require new human-relevant models. iPSCs allow addressing gene-environment interactions. Therefore, we developed two 3D human neuronal models: (1) human iPSC-derived 3D mini-brains to recapitulate neurodevelopment and after maturation to selectively damage dopaminergic neurons by Parkinson agents, and (2) a homogeneous LUHMES 3D dopaminergic neuronal model, to study their molecular signatures and pathways.

There is a great need for new medicines with novel mechanisms that provide increased efficacy and improved safety. Recently approved medicines work in many different ways and phenotypic assays contributed to the identification of the many, different MMOAs. It is clear that phenotypic assays are an important tool to identify novel MMOAs and the corresponding medicines.

2:30 Small-Molecule Discovery for Diabetes: The Importance of Combining a Phenotypic Approach and Mechanism-of-Action Studies

Identifying small molecules that impact on pancreatic beta-cell survival and function could have a great impact on developing new diabetes therapies. Taking a phenotypic cell-based approach can help speed this process. However, a major bottleneck in the process is determining the mechanism of action of novel compounds. Here, I will discuss our approaches to small-molecule discovery in the beta cell, as well as current efforts to streamline the process of understanding mechanistic activity.

Efforts to develop more effective cancer therapies may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including cancer stem cells and supportive interactions with the malignant microenvironment. Primary cancer cells were cultured with stromal cells, as were primary normal cells, revealing cancer-specific stem-cell dependencies by small-molecule screening.

For successful identification of high quality hits, the host cellular system must be physiologically relevant. Primary and human-derived cell lines provide the most predictive biology but are difficult to modify. Designed for patients in cell therapy, MaxCyte flow electroporation is used in disease-specific research and high throughput screening with primary and human-derived cells. In this presentation, case studies on assay development with neuronal cells, hematopoietic cells, bronchial cells and tumor cell lines will be presented demonstrating the use of flow electroporation to create physiologically relevant assays.

Microscopy images contain rich information about the state of cells, tissues and organisms. Our laboratory extracts patterns of morphological perturbations (“signatures”) from images to identify similarities and differences between chemical or genetic treatments, with the ultimate goal to identify the causes and potential cures of disease. Using model systems that are more and more physiologically relevant, we are developing assays and accompanying algorithms to extract multi-parametric morphological fingerprints of cell populations.

Binding of a small molecule to its target domain is a prerequisite of biological activity. By interrogating existing target-ligand pairs covering the known ligandable proteome, we can predict candidate target-ligand pairs for phenotypic screening hits. An alternative to this method is using purified protein libraries representing ligandable domains to screen for protein-target domain interactions in a label-free format. This talk will focus on implementation of both technologies for generating and validating target-ligand hypotheses for bioactive compounds emerging from phenotypic screens.

5:35 Repurposing Drug Screens to Elucidate New Drug Targets and Pathways for Infectious Disease and Cancer

NCATS has established a unique approved drug collection containing 4,265 compounds. Using this library, we have screened against several disease models, including malaria gametocytes, antibiotic-resistant bacteria and drug-resistant ovarian cancer. Compared to target-based screens, these phenotypic-based screens are more disease relevant, offering additional biological complexity. Not only were potent candidates for further drug development identified, but these screens also revealed novel molecular targets and pathways that increased insight into these disease processes.

Native cell types with relevance to drug discovery in cardiac regeneration and islet health are being used by AstraZeneca to develop assays with improved physiological relevance over traditional approaches. Development and application of phenotypic assays using cellular systems utilizing the iPS technology, precise genome editing (PGE) technologies and primary human cells will be discussed. The presentation will use case studies to demonstrate both the advantages and hurdles of these approaches to identify novel targets, pathways or mode of action.

Disorders with complex genetics, such as schizophrenia and bipolar disorder, provide a significant challenge for development of novel therapeutics through target-based methods. The lack of reliable and valid disease-related cellular features makes phenotypic screening difficult as well. This presentation will cover some approaches that are being explored with human neurons differentiated from patient iPSCs in order to identify phenotypic signatures that can be used for signature-based screens.

Development of technology platforms that can be used to perform compound screens against patient-specific human induced pluripotent stem cell (hiPSC)-derived cell types with relatively high throughput will be essential to realize the potential that these cells hold for disease modeling and drug discovery. Towards this goal, we have been working to develop a standardized battery of assays against which hiPSC-derived neurons can be screened for specific phenotypes. Our results demonstrate the feasibility of performing higher throughput drug screens on hiPSC-derived neurons and establish platforms for future screens using patient specific cells.

This presentation will cover a collection of scientific vignettes (i.e., lessons) based on in-house experience with phenotypic drug discovery and touching on a broad range of topics including designing the best phenotypic assays, primary cells and donor-to-donor variability analysis, hit triage and validation, use and misuse of chemogenomics libraries, toxicity and safety derisking of PDD hits.

Recent advances in the fields of biology, chemistry, proteomics and screening technology have greatly improved our chances of identifying underlying protein targets. We will highlight progress on all these fronts as they relate to the discovery of mechanisms and chemical matter, derived from an ultra-high throughput phenotypic screen, which induces latent HIV expression in infected cells.

Sickle cell disease (SCD) is one of the most common genetic diseases and a major source of morbidity and mortality worldwide. Increasing fetal hemoglobin (HbF) has long been a therapeutic goal in management of SCD. This presentation will cover the strategy and outcome of phenotypic screens for HbF inducers. I will discuss the importance of setting up physiologically relevant cellular models in hit identification, the challenges and learnings from screens using primary human cells and high-content data handling and analysis. The value of using annotated chemical libraries in phenotypic screening and the discovery of novel chemical modulators will also be presented.